Construction And Application Of Neuronal Specific Gene Expression System In C. Elegans

C. elegans is the most fruitful experimental model to advance our understanding of the molecular mechanisms of development and the function of nervous system. It has become such a pin-up animal for scientists.Although it’s apparent simplicity, the nematode worm C. elegans has been a priceless model for biological research, principally in the functional characterization of uni ue target genes that have been identified using genomics technologies. C. elegans and advanced organisms along with the simplicity and cost-effectiveness of the culture, make it as an effective in vivo model which is amenable to whole-organism for high productivity compound screening and extensive target validation. C. elegans nervous system is very simple and adult has only302neurons, which can be divided into motor neuron, sensory neuron and interneuron. Thus, we used C. elegans as a object of study in my work. Here Ⅰ combined the gateway technology and site-specific recombinase system (Cre/loxP, FLP/FRT), with confocal laser scanning technique in order to label C. elegans neurons. We chose the promoters with intersections, so that we can label neurons by different colors with different fluorescent proteins driven by different promoters express. In addition we also created a combined system which enrolled intersection of promoters and site-specific recombinase system (CreLoxP, FLP/FRT), to label single neurons. We have constructed vectors containing FLP/FRT or Cre//oxP specific recombination system to explore two specific promoter-driven neuron expression systems. Also we have constructed vectors containing (Cre/loxP and FLP/FRT) specific recombination system to explore three specific promoter-driven neuron expression systems. Moreover; we completed construction of promoter library based on Gateway technology, so that the promoters and coding regions can be quickly matched and mixed which would greatly facilitate/promote the downstream research of C. elegans and realizing spatial and temporal gene expression. Inducible neuron-specific gene expression system will not only greatly facilitate the study of function and behavior of C. elegan, it can also establish an solid foundation for the three-dimensional reconstruction of neuron in C. elegans.On the other hand, we also found some ways for constructing plasmids through marking the specific neurons have been found out, particularly the combination of the restriction digestion method and multisite gateway technique, not only showing the high effectiveness and seamless ligation in restriction digestion reaction, but also making of repeated use in multisite gateway reaction. At the same time, this method can also being used for genes’ conditional expression, to further study the neurons" and genes’ function in the behavior of C.elegan.In this regard, we have seccessfuly labelled the sensory neuron ASH and ASI, by intersection of two promoters Psra-6and gpa-11P, rab-3P and gba-4P respectively. And we also succeded in labelling the neuron ASI by intersection of three promoter’s rab-3P and we have used the second promoter and the third of the same (gba-4P) that can be expressed in single neurons ASI. Therefore, further functional studies of nervous system of C. elegans can be continued using this system.